Prior investigators recognized that mixed cell suspensions can be analyzed for the presence or absence of cell populations and for quantitative information concerning the number of specific types of cells present in the sample. This information is useful in the diagnosis of disease and in the monitoring of the effectiveness of therapy for certain disease conditions. Prior investigators further recognized that detectable differences in cell population responses to conditions imposed upon the mixed cell suspension can be advantageously exploited to signal the presence or absence of specific cell populations. These detectable differential responses can also be used to generate signals which are unique to a single cell population, enabling enumeration of that cell population. The mixed cell systems in which differential response techniques have been studied include physiological fluids such as blood, blood plasma, bone marrow, semen and organ cell suspensions as well as cell culture media, including plant cell suspensions. Such response techniques range from differential cell staining in the presence of an added dye to failure of one or more cell populations to survive an imposed stress such as brief or prolonged exposure to an extreme in temperature, pH, tonicity or chemical microenvironment.
The selection of an imposed condition by which to generate a differential response from a mixed cell suspension is typically a compromise. The inherent similarities between cell populations present in the cell suspension inadvertently lead to some partial response from the untargeted cell population which adversely affects the quality of the information obtained in all such types of differential response techniques.
Mammalian blood is one of the more thoroughly studied mixed cell suspensions which is analyzed utilizing various differential response techniques-including a differential cell-population-survivorship response. The cell populations present in whole blood are the erythrocytes (E or red cells) whose concentration in mammals is in millions per microliter; the anucleate thrombocytes (T or platelets) whose concentrations in mammals is generally in tens to hundreds of thousands per microliter; and the nucleated leukocytes (or white cells) whose concentration in mammals generally ranges from hundreds per microliter to tens of thousands per microliter and sometimes beyond (for any sub-population of leukocytes). Leukocytes in peripheral blood of normal mammals are further classified into five major types or subpopulations: the smaller lymphocytes (L), and the larger neutrophils (Ne), eosinophils (Eo), basophils (Ba) and monocytes (Mo).
These blood cell populations are visible in FIG. 1 in which the neutrophils, eosinophils and basophils are collectively identified as granulocytes (G). All five major leukocyte subpopulations are shown in FIG. 6.
One of the major distinctions between erythrocytes and leukocytes is that, in health, it is relatively simple to identify erythrolytic agents which selectively destroy (or lyse away) the erythrocytes while leaving the typical leukocytes substantially intact. Enormous differences in cytoplasmic structure account for this differing survivorship response. When a leukocyte population in solution is presented with physico-chemical conditions which cause all the erythrocytes in the whole blood sample to exceed this critical hemolytic threshold, these erythrocytes in the whole blood sample will have been lysed away by a rapid cytolytic decay process which is mainly an interaction of osmotic, oncotic and surface membrane phenomena. It is predicted that, usually, all of the leukocyte subpopulations will also commence their own lytic decay processes. However, unlike mature erythrocytes, leukocytes (including thrombocytes) have a large amount of readily recruitable redundant internalized cell membrane. Under appropriate conditions, this membrane material can be externalized so that leukocytes share many of the characteristics of a soap bubble under inflation. Those soap bubbles which can still be inflated further still have readily recruitable, spare (non-surface) soap molecules in reserve. Erythrocytes which are more mature than reticulocytes no longer have any redundant surface material. As a result, by comparison with erythrocytes, the rate at which the members of the leukocyte populations reach their critical hemolytic threshold is generally so slow that, under many temporal conditions of analysis, leukocyte populations can be viewed as effectively lyse-resistant.
For over a century, this differing cell-survivorship response has been exploited haphazardly to permit categorization and enumeration of leukocytes present in a whole blood sample. It is evident from FIG. 1 that if the erythrocytes are simply lysed away, while the leukocytes and thrombocytes are preserved, then it is no longer necessary to process over ten thousand erythrocytes for every single member of the less numerous leukocyte subpopulations. This reduced processing burden greatly shortens the time (and cost) for analyzing a whole blood sample. However, exploitation of this differing cell-survivorship response to lytic agents in the century old techniques has very real limitations. A compromise must be struck between the use of strongly erythrolytic conditions which effect complete erythrolysis (at the cost of some leukocytes) and the use of less strongly erythrolytic conditions to leave the leukocytes unaffected (at the cost of impairing leukocyte counting because of the presence of unlysed interfering erythrocytes). Over the last one hundred fifty years, we have not found an optimum compromise condition which simultaneously addresses these conflicting responses in both health and disease.
Consequently, the cost-effective and rapid erythrolytic procedures advantageous for automation in human and veterinary clinical use are also intrinsically leukolytic (white cell and platelet lysing). The accurate categorization and enumeration of leukocyte subpopulations can thereby be compromised. The leukolytic activity accompanying erythrolysis degrades the clinical categorization and enumeration of the leukocyte subpopulations since the accuracy of the count is greatly reduced. Therefore, it is one object of the present invention to provide a method for categorizing and enumerating leukocyte subpopulations which method accounts for (and corrects the enumeration for) the inherent leukolytic activity of commonly used erythrolytic agents and conditions.